Isolation and characterization of kinetoplast DNA from bloodstream form of Trypanosoma brucei

We have used restriction endonucleases PstI, EcoRI, HapII, HhaI, and S1 nuclease to demonstrate the presence of a large complex component, the maxi-circle, in addition to the major mini-circle component in kinetoplast DNA (kDNA) networks of Trypanosoma brucei (East African Trypanosomiasis Research Organization [EATRO] 427). Endonuclease PstI and S1 nuclease cut the maxi-circle at a single site, allowing its isolation in a linear form with a mol wt of 12.2 x 10(6), determined by electron microscopy. The other enzymes give multiple maxi-circle fragments, whose added mol wt is 12-13 x 10(6), determined by gel electrophoresis. The maxi-circle in another T. brucei isolate (EATRO 1125) yields similar fragments but appears to contain a deletion of about 0.7 x 10(6) daltons. Electron microscopy of kDNA shows the presence of DNA considerably longer than the mini-circle contour length (0.3 micron) either in the network or as loops extending from the edge. This long DNA never exceeds the maxi-circle length (6.3 microns) and is completely removed by digestion with endonuclease PstI. 5-10% of the networks are doublets with up to 40 loops of DNA clustered between the two halves of the mini-circle network and probably represent a division stage of the kDNA. Digestion with PstI selectively removes these loops without markedly altering the mini-circle network. We conclude that the long DNA in both single and double networks represents maxi-circles and that long tandemly repeated oligomers of mini-circles are (virtually) absent. kDNA from Trypanosoma equiperdum, a trypanosome species incapable of synthesizing a fully functional mitochondrion, contains single and double networks of dimensions similar to those from T. brucei but without any DNA longer than mini-circle contour length. We conclude that the maxi-circle of trypanosomes is the genetic equivalent of the mitochondrial DNA (mtDNA) of other organisms.     

The kinetoplast DNA of Trypanosoma equiperdum

We have analyzed the kinetoplast DNA for Trypanosoma equiperdum (American Type Culture Collection 30019) and two dyskinetoplastic strains derived from it. The DNA networks from the kinetoplastic strain are made up of catenated mini-circles and maxi-circles, like the networks from the closely-related Trypanosoma brucei. The mini-circles of T. equiperdum lack the pronounced sequence heterogeneity of T. brucei mini-circles, as shown by the fragment distribution of restriction digests and by the predominance of well-matched duplexes in electron micrographs of renatured DNA. The electrophoretic analysis of kinetoplast DNA digested with various restriction endonucleases shows the maxi-circle of T. equiperdum to consist of circular DNA molecules of 8.4 x 10(6) daltons, without size or sequence heterogeneity or repetitious segments. A comparison of the sequence by restriction endonuclease fragmentation and hybridization shows extensive sequence homology. The size difference between both maxi-circles is due to the deletion of one continuous segment of 5.10(6) daltons. In the two dyskinetoplastic strains, we cannot detect DNA sequences that hybridize with kinetoplast DNA from T. brucei or from the kinetoplastic strain of T. equiperdum. In one of these strains, a 'low-density' DNA fraction contained a simple sequence DNA, cleaved by restriction endonuclease HindIII into fragments of 180 base-pairs and multimers of this. The relation of this DNA to kinetoplast DNA, if any, is unknown.     

The structure of kinetoplast DNA. 1. The mini-circles of Crithidia lucilae are heterogeneous in base sequence.

We have analysed limit digests of mini-circles from kinetoplast DNA of Crithidia luciliae by gel electrophoresis. Endonucleases HapII and AluI cut the circles into at least 37 and 21 fragments, respectively, and leave no circles intact. In both cases the added molecular weights of the fragments, estimated from mobility in gels, exceeds 18 X 10(6), i.e. more than 12 times the molecular weight of the mini-circle DNA. Endonucleases HindII + III, EcoRI and HpaI cut only part of the circles. These results show that the mini-circles are heterogeneous in base sequence. Different sequence classes are present in different amounts. DNA-DNA renaturation analysis of mini-circle DNA yields a complexity of about 3 X 10(6), i.e. twice the molecular weight on one mini-circle. The delta tm of native and renatured duplexes is about 1 degree C, showing that the sequence heterogeneity is a micro-heterogeneity. Electron microscopy, gel electrophoresis and sedimentation analysis show that the circles that are not cut by endonucleases HindII + III remain catenated in very large associations. These associations lack the 'rosette' structures and the long edge loops characteristic of intact kinetoplast DNA. This suggests that the mini-circle classes cut by endonucleases HindII + III are present throughout the network and that the maxi-circle component of the network (see accompanying paper) is not essential to hold the network together. Prolonged electrophoresis on 1.5% or 2% agarose gels resolves the open mini-circles into three and linearized mini-circles into four bands, present in different amounts. We conclude that the mini-circles are also heterogeneous in size, the difference in size between the two extreme size classes being 4% of the contour length. Digestion with endonuclease HapII shows that at least three out of these four bands differ in sequence. Possible mechanisms that could account for the micro-heterogeneity in sequence of mini-circles are discussed.     

Analysis by electron microscopy of the variable segment in the maxi-circle of kinetoplast DNA from Trypanosoma brucei

The 20.5-kbp maxi-circle from the kinetoplast DNA of Trypanosoma brucei contains a 5-kbp segment which is not cut by most restriction endonucleases and which varies in size in closely-related trypanosome strains (Borst, P., Fase-Fowler, F., Hoeijmakers, J.H.J. and Frasch, A.C.C. (1980) Biochim. Biophys. Acta 610, 197–210). We have now analysed partial denaturation maps of the linearized maxi-circles by electron microscopy and find that the variable segment is not more AT-rich than the remainder of the maxi-circle. Early denaturation begins at two separate regions of the maxi-circle outside the variable region and one of these corresponds with the position of the gene for the large (12 S) ribosomal RNA. Denaturation-renaturation of maxi-circles leads to the formation of partially mismatched duplexes that look like underwound loops in electron micrographs. These loops are only found in the variable region and they vary in size and appearance. Under our renaturation conditions single-stranded maxi-circle DNA is devoid of secondary structure and this suggests that the underwound loops arise by misalignment of straight tandem repeats in the DNA. We have also analysed heteroduplexes between maxi-circles from two closely related T. brucei strains that differ by 1 kbp in the size of their variable segment. Most molecules had no underwound loops and contained mismatched regions in the variable segment only. The appearance of these regions is diverse, varying from fully duplex with two single-stranded loops to molecules with a heterogeneous array of smaller loops. The total size of single-stranded DNA in the heteroduplexes may be as high as 1.2 μm, i.e., a factor 4 higher than the size difference between the heteroduplex partners. We conclude that the variable region consists of imperfect tandem repeats of a sequence that evolves rapidly. This region might contain the origin of maxi-circle replication.     

Discontinuously synthesized mRNA from Trypanosoma brucei contains the highly methylated 5' cap structure, m7GpppA*A*C(2'-O)mU*A

Trypanosoma brucei mRNA is discontinuously synthesized via the 5' addition of a "mini-exon" sequence. The mini-exon-specific cap structure was purified from a complete RNase T2 and phosphatase digest of in vivo 32P-labeled poly(A)+RNA. The purified cap structure was sequenced by a series of partial and complete enzymatic digests by nuclease P1 and venom phosphodiesterase. This approach demonstrated that the T. brucei mini-exon cap structure consists of N7-methylguanosine linked in a conventional 5'-5' triphosphate bond to five nucleotides, in the sequence A*A*C(2'-O)mU*A (asterisks denote modifications that were not fully characterized in this work). 2'-O-methylations and other modifications appear to be present in this novel cap structure, which could have a functional role in the metabolism of the mini-exon.     

A comparison of Glossina morsitans centralis originating from Tanzania and Zambia, with respect to vectorial competence for pathogenic Trypanosoma species, genetic variation and inter-colony fertility

wo laboratory strains of Glossina morsitans centralis originating from different fly-belts (one from Singida, in Tanzania, and the other from Mumbwa, in Zambia) were compared with respect to vectorial competence for pathogenic Trypanosoma species, genetic variation and inter-colony fertility. The vectorial competence of G.m.centralis of Tanzanian origin for Trypanosoma vivax and T.congolense is similar to, whereas for T.brucei brucei it is lower than the colony of Zambian origin. Nevertheless, these two laboratory strains of G.m.centralis showed levels of susceptibility to the three pathogenic Trypanosoma species which were much greater than previously observed in laboratory colonies of other Glossina species. Electrophoresis of fifteen enzymes revealed that the two colonies differ significantly in allele frequencies at only three loci that are relatively close together on one of the autosomes. Hybridization experiments revealed that G.m.centralis from the two fly-belts are consubspecific.     

Improved separation of chromosome-sized DNA from Trypanosoma brucei, stock 427-60.

Separation of chromosome-sized DNA from the parasitic protozoan Trypanosoma brucei had previously resulted in the fractionation of DNA molecules that ranged in size from 50 kb up to roughly 1.5 Mb. The number of larger chromosomes and their size, accounting for 80% of the DNA of T. brucei remained unclear. We have now size separated these larger DNA molecules by pulsed field gel electrophoresis (PFG) and resolve a total of 20 bands, accounting for roughly 120 chromosomes, ranging in size from 50 kb up to the size of the largest, 5.7 Mb chromosome of Schizosaccharomyces pombe. Three different VSG gene expression sites were located to chromosomes of 430 kb, 1.5 Mb and 3 Mb, respectively. We have not been able to identify additional, previously cryptic DNA rearrangements, that could explain the activation or inactivation of the expression sites.     

The structure of kinetoplast DNA. 2. Characterization of a novel component of high complexity present in the kinetoplast DNA network of Crithidia luciliae.

1. Degradation of highly purified kinetoplast DNA (kDNA) networks with restriction endonucleases yields "extra" bands in agarose gels that are absent from digests of mini-circles. Each of the five endonucleases tested, i.e. AluI, HapII, EcoRI, Hsu and HindII + III, yields a unique set of "extra" bands. The "extra" bands consist of linear DNA; they are not mini-circle oligomers and their added molecular weight, calculated from mobility in gels, are around 2 X 10(7). Double digests with two restriction endonucleases yield a new set of "extra" bands, showing that the "extra" bands obtained with different enzymes are all derived from the same complex component of kDNA. In digests of 32P-labelled kDNA an average of 2.3% of the radioactivity is recovered in the "extra" bands. 2. Treatment of kDNA networks with the single-strand-specific S1 nuclease of Aspergillus oryzae preferentially releases a linear DNA with a molecular weight of 26 X 10(6), calculated from mobility in gels. We present evidence that the 'extra' bands obtained with restriction endonucleases are derived from this component. 3. DNA-DNA renaturation analysis of fragmented kDNA shows the presence of a minor complex component with a complexity of about 3 X 10(7), making up less than 10% of the total kDNA. 4. From these results we conclude that 3--5% of the kDNA consists of a homogeneous class of maxi-circles catenated in the mini-circle network. The molecular weight of these maxi-circles is about 26 X 10(6) and they contain a unique, non-repetitive, non-mini-circle nucleotide sequence. This component is a prime candidate for the true mitochondrial DNA of trypanosomes.     

Selective pressure can influence the resistance of Trypanosoma congolense to normal human serum

Resistance and sensitivity to normal human serum (NHS) of Trypanosoma congolense, a parasite believed to cause disease in animals only, were investigated in vivo as well as in vitro. Our results indicate that like Trypanosoma brucei, T. congolense can be grouped into three different phenotypes according to its resistance to NHS. Some strains are completely resistant to NHS, like Trypanosoma brucei gambiense and the resistant form of Trypanosoma brucei rhodesiense. Other strains show a very low degree of resistance comparable to the sensitive form of T. b. rhodesiense, and some are completely sensitive to NHS. Continuous passaging in mice in the presence or absence of NHS shows that the resistance and sensitivity of T. congolense can be reversed like in T. b. rhodesiense. Our data suggest that T. congolense might be able to infect man in regions where animals may serve as reservoirs for the infection.     

The tubulin genes of Trypanosoma cruzi

The organization of the alpha- and beta-tubulin genes in the genome of Trypanosoma cruzi have been analysed by Southern blotting using tubulin probes derived from Trypanosoma brucei. The tubulin array appears to be more complex in this organism than in other members of the same family. Some tubulin genes are tightly clustered in an alternating (alpha-beta)n array with a basic repeat unit length of 4.3 kb. However, other pairs of alternating alpha- and beta-tubulin sequences appear to be physically separated from the basic group. This finding indicates that the tubulin gene cluster present in T. cruzi is less perfectly conserved than in T. brucei. T. (Herpetosoma) rangeli is similar to T. (Schizotrypanum) cruzi in its tubulin gene organization whereas most of these genes are tandemly clustered in the genome of T. (Trypanozoon) evansi, with a basic repeat unit length of 3.6 kb as previously described for T. (Trypanozoon) brucei. Two overlapping recombinant clones containing T. cruzi tubulin sequences have been isolated from a genomic cosmid library of T. cruzi epimastigotes using the T. brucei tubulin probes. Partial sequencing of the T. cruzi beta-tubulin gene has confirmed its identity and shows more than 70% homology with the sea urchin, chicken and T. b. rhodesiense beta-tubulin reported gene sequences. Analysis of tubulin gene organization through the parasite life cycle does not show evidence of major rearrangements within the repeat unit. Several T. cruzi strains and cloned lines whilst sharing the 4.3-kb tubulin repeat unit, exhibited very variable tubulin gene organization with tubulin probes. These striking differences in the organization of this structural gene among T. cruzi strains and cloned lines suggest that the heterogeneity previously reported in parasite populations may be related to a very dynamic, diploid genome.     

Vector competence of Glossina palpalis gambiensis for Trypanosoma brucei s.l. and genetic diversity of the symbiont Sodalis glossinidius

Tsetse flies transmit African trypanosomes, responsible for sleeping sickness in humans and nagana in animals. This disease affects many people with considerable impact on public health and economy in sub-Saharan Africa, whereas trypanosomes' resistance to drugs is rising. The symbiont Sodalis glossinidius is considered to play a role in the ability of the fly to acquire trypanosomes. Different species of Glossina were shown to harbor genetically distinct populations of S. glossinidius. We therefore investigated whether vector competence for a given trypanosome species could be linked to the presence of specific genotypes of S. glossinidius. Glossina palpalis gambiensis individuals were fed on blood infected either with Trypanosoma brucei gambiense or Trypanosoma brucei brucei. The genetic diversity of S. glossinidius strains isolated from infected and noninfected dissected flies was investigated using amplified fragment length polymorphism markers. Correspondence between occurrence of these markers and parasite establishment was analyzed using multivariate analysis. Sodalis glossinidius strains isolated from T. brucei gambiense-infected flies clustered differently than that isolated from T. brucei brucei-infected individuals. The ability of T. brucei gambiense and T. brucei brucei to establish in G. palpalis gambiensis insect midgut is statistically linked to the presence of specific genotypes of S. glossinidius. This could explain variations in Glossina vector competence in the wild. Then, assessment of the prevalence of specific S. glossinidius genotypes could lead to novel risk management strategies.     

DNA metabolism and genetic diversity in Trypanosomes

Trypanosomes are protozoan parasites that cause major diseases in humans and other animals. Trypanosoma brucei and Trypanosoma cruzi are the etiologic agents of African and American Trypanosomiasis, respectively. In spite of large amounts of information regarding various aspects of their biology, including the essentially complete sequences of their genomes, studies directed towards an understanding of mechanisms related to DNA metabolism have been very limited. Recent reports, however, describing genes involved with DNA recombination and repair in T. brucei and T. cruzi, indicated the importance of these processes in the generation of genetic variability, which is crucial to the success of these parasites. Here, we review these data and discuss how the DNA repair and recombination machineries may contribute to strikingly different strategies evolved by the two Trypanosomes to create genetic variability that is needed for survival in their hosts. In T. brucei, two genetic components are critical to the success of antigenic variation, a strategy that allows the parasite to evade the host immune system by periodically changing the expression of a group of variant surface glycoproteins (VSGs). One component is a mechanism that provides for the exclusive expression of a single VSG at any one time, and the second is a large repository of antigenically distinct VSGs. Work from various groups showing the importance of recombination reactions in T. brucei, primarily to move a silent VSG into an active VSG expression site, is discussed. T. cruzi does not use the strategy of antigenic variation for host immune evasion but counts on the extreme heterogeneity of their population for parasite adaptation to different hosts. We discuss recent evidence indicating the existence of major differences in the levels of genomic heterogeneity among T. cruzi strains, and suggest that metabolic changes in the mismatch repair pathway could be an important source of antigenic diversity found within the T. cruzi population.     

Kinetoplast DNA of Trypanosoma brucei: Physical map of the maxicircle

Trypanosoma brucei maxicircle DNA in kinetoplast DNA (kDNA) networks was characterized with restriction endonucleases. The data allow the construction of a circular map of a 22.2-kb molecule. Based on these and previous data each T. brucei kDNA network contains about 45 maxicircles which probably have the same sequence. The maxicircle of strain 164 used in this study was slightly larger and had three EcoRI sites compared to two found in other strains. Fragments generated by digestion with BamHI were largely singly cleaved maxicircles that had a density of 1.681 g/cm³ compared to 1.693 g/cm³ for the intact network. This suggests that maxicircles have a higher A + T content than minicircles. Minicircles in the kDNA network were also characterized with restriction endonucleases. Each enzyme cleaved a specific subset of minicircles from the network. However, no single restriction endonuclease or combination of up to three of these enzymes cleaved all molecules in the network. These results are consistent with earlier results of renaturation kinetic experiments and indicate that there are many different sequence classes of mini-circle DNA.     

Evolution of codon usage and base contents in kinetoplastid protozoans

In this study we analyze and compare the trends in codon usage in five representative species of kinetoplastid protozoans (Crithidia fasciculata, Leishmania donovani, L. major, Trypanosoma cruzi and T. brucei), with the purpose of investigating the processes underlying these trends. A principal component analysis shows that the G+C content at the third codon position represents the main source of codon-usage variation, both within species (among genes) and among species. The non- Trypanosoma species exhibit narrow distributions in codon usage, while both Trypanosoma species present large within-species heterogeneity. The three non-Trypanosoma species have very similar codon-usage preferences. These codon preferences are also shared by the highly expressed genes of T. cruzi and to a lesser degree by those of T. brucei. This leads to the conclusion that the codon preferences shared by these species are the ancestral ones in the kinetoplastids. On the other hand, the study of noncoding sequences shows that Trypanosoma species exhibit mutational biases toward A + T richness, while the non- Trypanosoma species present mutational pressure in the opposite direction. These data taken together allow us to infer the origin of the different codon-usage distributions observed in the five species studied. In C. fasciculata and Leishmania, both mutational biases and (translational) selection pull toward G + C richness, resulting in a narrow distribution. In Trypanosoma species the mutational pressure toward A + T richness produced a shift in their genomes that differentially affected coding and noncoding sequences. The effect of these pressures on the third codon position of genes seems to have been inversely proportional to the level of gene expression.